Project Summary RNA-binding proteins are essential components of numerous large complexes that carry out fundamental processes including transcription, splicing, and DNA repair. Many RNA-binding proteins possess low- complexity domains that are critical to normal RNA-processing functions, but also drive aberrant protein assembly in various types of neurodegenerative disease and cancer. The structure and function of these low complexity domains remain poorly characterized, especially in the context of disease. Fused in Sarcoma (FUS) is one of twenty-nine distinct human RNA-binding proteins that contain an essential putatively unstructured low-complexity domain with unusually low charged residue composition and a high frequency of aromatic amino acids. The low-complexity domain of FUS is thought to facilitate interactions in normal RNA metabolism by forming dynamic associations, enabling tunable, reversible spatial clustering. Yet, excessive self- association between FUS low-complexity domains is believed to result in the formation of pathological neuronal inclusions in sub-types of amyotrophic lateral sclerosis and frontotemporal dementia, which are irreversible neurodegenerative diseases that lack effective treatments. Moreover, fusion of the FUS low- complexity domain to certain DNA-binding domains through chromosomal translocations results in uncontrolled gene expression leading to a family of aggressive cancers of childhood. The structures of FUS assemblies and the normal mechanisms that prevent disease-associated aggregates and complexes are currently unknown because they are invisible to traditional techniques in structural biology. However, recent technical advances now enable visualization of dynamic assemblies of FUS with residue-level resolution. This project will apply advanced nuclear magnetic resonance spectroscopy, molecular simulation, and cell models of FUS-associated diseases to 1) map the structure and molecular contacts of the low-complexity domain of FUS along its assembly pathway, 2) elucidate the molecular details and consequences of disease-associated mutations and posttranslational modifications of the FUS low-complexity domain, and 3) determine atomic details of the complex formed between self-assembled FUS and the C-terminal domain (CTD) of RNA polymerase II; and evaluate how altering the interaction between FUS and CTD, by modifying the FUS low-complexity domain, affects transcriptional activation and cancerous transformation potential. These studies of FUS assembly will provide necessary structure/function information on future pharmacological targets for inhibiting pathological protein associations in types of amyotrophic lateral sclerosis, frontotemporal dementia, leukemia, and sarcoma. Furthermore, because FUS is only one of many essential RNA-binding proteins containing aggregation-prone low complexity domains, the results of the project will serve as the foundation for understanding the interactions of an entire class of proteins and for correcting their dysfunctions in disease.